Consensus in silico computational modelling of the p22^phox subunit of the NADPH oxidase

Abstract

The p22^phox protein is an essential subunit of the cytochrome b₅₅₈ of the NADPH oxidase (Nox) complex which by generating reactive oxygen species (ROS) plays important role in regulating cellular function. p22^phox stabilises the Nox enzyme, assists in catalytic core maturation and in the meantime provides an anchoring site for cytosolic regulatory subunits to bind. However, the protein structure of the p22^phox is still uncertain. In this study we use an in silico computational bioinformatic approach to produce a consensus 3-dimensional model of the p22^phox. Based on published protein sequence data of human p22^phox and by using transmembrane specific protein prediction algorithms, we found that p22^phox consists of two domains: an N-terminal transmembrane domain (124 a.a.) and a C-terminal cytoplasmic domain (71 a.a.). In its predicted most stable form, p22^phox contains three transmembrane helices leading to an extracellular N-terminus and an extensive (39 a.a.) extracellular loop between helices 2 and 3. Furthermore, we locate the cytosolic domain phosphorylation site at threonine₁₄₇ which literature shows is capable of priming the p22^phox, in order to accept its binding partners. Our results are consistent with the biological characterisation of p22^phox derived from experiments using specific antibody or genetic manipulation. Our 3-D protein model provides insights into the biological function of p22^phox and cytochrome b₅₅₈, and can be used as tool to investigate the regulatory mechanism of Nox isoforms.

Introduction

The phagocytic type nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (also called Nox2) is a membrane bound enzyme complex that produces superoxide (O₂⁻) as an essential precursor for other reactive oxygen metabolites critical for oxygen-dependant microbiocidal activity and redox-sensitive cellular processes (Sumimoto, 2008). The physiological importance of Nox2 is demonstrated by the rare genetic disorder chronic granulomatous disease (CGD), where mutations in any regulatory subunit cause defective oxidase activity and the inability of phagocytes to destroy pathogenic microorganisms (Kuhns et al., 2010). The activated human Nox2 isoform is composed of the heterodimeric membrane associated cytochrome b₅₅₈, consisting of the catalytic subunit gp91^phox (a.k.a. Nox2) and p22^phox, and regulatory cytosolic subunits (p47^phox, p67^phox, p40^phox and Rac1/2). Nox2 activation and subsequent ROS production is well defined in the literature and occurs when cytosolic components assemble at the membrane anchored by the p22^phox subunit (Li and Shah, 2004).

Although Nox-derived reactive oxygen species (ROS) are crucial for normal homeostasis, excessive ROS production due to Nox activation has been found to be associated with oxidative stress-related pathological complications, including coronary heart disease, hypertension, atherosclerosis and type II diabetes, as well as neurological and inflammatory diseases (Lambeth et al., 2008). To date, 7 members of the Nox/DUOX family of ROS producing enzymes have been discovered with an extensive tissue distribution throughout the body, where Nox1–4 are found interacting with p22^phox within membranes (Bedard and Krause, 2007).

Despite a wealth of knowledge pertaining to the functional role of the NADPH oxidase, there are currently no complete x-ray crystal structures for any Nox isoform or p22^phox. There are reports containing partial x-ray structures of particular domains of the p22^phox, e.g. the proline rich region of the p22^phox bound to the activated p47^phox src homology 3 (SH3) domains (Groemping et al., 2003). Importantly, a protein's structure dictates its overall function and there is only one previous modelling study which looked to identify important aspects of the cytochrome b₅₅₈, focusing on the catalytic gp91^phox subunit and did not include any other oxidase subunits (Taylor et al., 1993). In silico protein modelling is the accumulation of decades of structural biology research and is becoming an important area of interest for functional biologists in studying the p22^phox (Taylor et al., 2011). The anchoring subunit has been shown as an important partner for maturation of the catalytic subunit, despite a lack of understanding of the respective mechanistic processes. Moreover, previous analysis of the p22^phox protein sequence failed to identify important structural clues in relation to its overall shape, causing conflicting opinions within the literature (Dahan et al., 2002, Groemping and Rittinger, 2005). Furthermore, being a transmembrane (TM) protein, the p22^phox contains a large number of hydrophobic residues that compliment the membranes interior. However, lack of an appropriate topology model or x-ray crystal structure limits structural research relating to the manipulation of p22^phox as a potential therapeutic target in disease. Importantly, identification of crucial structural aspects allows us to develop new therapies, as well as to better understand disease mechanisms (Li et al., 2008).

The role of p22^phox in the production of an active Nox complex is important in the regulation of normal homeostasis. Currently there is a lack of consensus between the accepted topology for the p22^phox and therefore its role in overall Nox stability. In this study we present a novel model of the p22^phox subunit based on a comparison of computational homology modelling methods resulting in a consensus model which satisfies the current understanding of p22^phox biology.